Intracellular Ca2+ storage in acidocalcisomes of Trypanosoma cruzi.
TL;DR: Results are consistent with the presence of a Ca2+/H+ exchange system in an acidic vacuole, which is named the 'acidocalcisome' and appears to be a unique organelle present in trypanosomatids.
Abstract: The use of digitonin to permeabilize the plasma membrane of Trypanosoma cruzi allowed the identification of a non-mitochondrial nigericin- or bafilomycin A1-sensitive Ca(2+)-uptake mechanism. Proton uptake, as detected by ATP-dependent Acridine Orange accumulation, was also demonstrated in these permeabilized cells. Under these conditions Acridine Orange was concentrated in abundant cytoplasmic round vacuoles. This latter process was inhibited (and reversed) by bafilomycin A1, nigericin and NH4Cl in different stages of T. cruzi. Ca2+ released Acridine Orange from permeabilized cells, suggesting that the dye and Ca2+ were being accumulated in the same acidic compartment and that Ca2+ was taken up in exchange for protons. Addition of bafilomycin A1 (5 microM), nigericin (1 microM) or carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; 1 microM) to fura 2-loaded epimastigotes increased their intracellular Ca2+ concentration ([Ca2+]i). Although this effect was more noticeable in the presence of extracellular Ca2+, it was also observed in its absence. Addition of NH4Cl (10-40 mM) to different stages of T. cruzi, in the nominal absence of extracellular Ca2+ to preclude Ca2+ entry, increased both [Ca2+]i in fura 2-loaded cells, and intracellular pH (pHi) in 2',7'-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein acetoxymethyl ester (BCECF)-loaded cells. Treatment of the cells with the Ca2+ ionophore ionomycin under similar conditions (nominal absence of extracellular Ca2+) resulted in an increase in [Ca2+]i and a significantly higher increase in [Ca2+]i after addition of NH4Cl, nigericin or bafilomycin A1, all agents which increase the pH of acidic compartments and make ionomycin more effective as a Ca(2+)-releasing ionophore. Similar results were obtained when the order of additions was reversed. Taking into account the relative importance of the ionomycin-releasable and the ionomycin plus NH4Cl-releasable Ca2+ pools, it is apparent that most of the Ca2+ stored in different stages of T. cruzi is present in the acidic compartment thus identified. Taken together, these results are consistent with the presence of a Ca2+/H+ exchange system in an acidic vacuole, which we have named the 'acidocalcisome' and which appears to be a unique organelle present in trypanosomatids.
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TL;DR: There is extensive evidence that the remarkable properties of Poly P as a polyanion have made it suited for a crucial role in the emergence of cells on earth, and this review pays particular attention to the enzyme, polyphosphate kinase 1, responsible for Poly P synthesis and highly conserved in many bacterial species, including 20 or more of the major pathogens.
Abstract: Inorganic polyphosphate (Poly P) is a polymer of tens to hundreds of phosphate residues linked by "high-energy" phosphoanhydride bonds as in ATP. Found in abundance in all cells in nature, it is unique in its likely role in the origin and survival of species. Here, we present extensive evidence that the remarkable properties of Poly P as a polyanion have made it suited for a crucial role in the emergence of cells on earth. Beyond that, Poly P has proved in a variety of ways to be essential for growth of cells, their responses to stresses and stringencies, and the virulence of pathogens. In this review, we pay particular attention to the enzyme, polyphosphate kinase 1 (Poly P kinase 1 or PPK1), responsible for Poly P synthesis and highly conserved in many bacterial species, including 20 or more of the major pathogens. Mutants lacking PPK1 are defective in motility, quorum sensing, biofilm formation, and virulence. Structural studies are cited that reveal the conserved ATP-binding site of PPK1 at atomic resolution and reveal that the site can be blocked with minute concentrations of designed inhibitors. Another widely conserved enzyme is PPK2, which has distinctive kinetic properties and is also implicated in the virulence of some pathogens. Thus, these enzymes, absent in yeast and animals, are novel attractive targets for treatment of many microbial diseases. Still another enzyme featured in this review is one discovered in Dictyostelium discoideum that becomes an actin-like fiber concurrent with the synthesis, step by step, of a Poly P chain made from ATP. The Poly P-actin fiber complex, localized in the cell, lengthens and recedes in response to metabolic signals. Homologs of DdPPK2 are found in pathogenic protozoa and in the alga Chlamydomonas. Beyond the immediate relevance of Poly P as a target for anti-infective drugs, a large variety of cellular operations that rely on Poly P will be considered.
610 citations
TL;DR: Acidocalcisomes were first described in trypanosomatids and have been characterized in most detail in these species, and are linked with several functions, including storage of cations and phosphorus, polyphosphate metabolism, calciumHomeostasis, maintenance of intracellular pH homeostasis and osmoregulation.
Abstract: Recent work has shown that acidocalcisomes, which are electron-dense acidic organelles rich in calcium and polyphosphate, are the only organelles that have been conserved during evolution from prokaryotes to eukaryotes Acidocalcisomes were first described in trypanosomatids and have been characterized in most detail in these species Acidocalcisomes have been linked with several functions, including storage of cations and phosphorus, polyphosphate metabolism, calcium homeostasis, maintenance of intracellular pH homeostasis and osmoregulation Here, we review acidocalcisome ultrastructure, composition and function in different trypanosomatids and other organisms
424 citations
TL;DR: Characteristics of the platelet dense granules, together with their known acidity and high density, are similar to those of acidocalcisomes (volutin granules
405 citations
TL;DR: It is demonstrated that microneme discharge is regulated by parasite cytoplasmic free Ca2+ and that the micronemal contents, including the MIC2 adhesin, are released through the extreme apical tip of the parasite.
Abstract: Apicomplexan parasites, including Toxoplasma gondii, apically attach to their host cells before invasion. Recent studies have implicated the contents of micronemes, which are small secretory organelles confined to the apical region of the parasite, in the process of host cell attachment. Here, we demonstrate that microneme discharge is regulated by parasite cytoplasmic free Ca2+ and that the micronemal contents, including the MIC2 adhesin, are released through the extreme apical tip of the parasite. Microneme secretion was triggered by Ca2+ ionophores in both the presence and the absence of external Ca2+, while chelation of intracellular Ca2+ prevented release. Mobilization of intracellular calcium with thapsagargin or NH4Cl also triggered microneme secretion, indicating that intracellular calcium stores are sufficient to stimulate release. Following activation of secretion by the Ca2+ ionophore A23187, MIC2 initially occupied the apical surface of the parasite, but was then rapidly treadmilled to the posterior end and released into the culture supernatant. This capping and release of MIC2 by ionophore-stimulated tachyzoites mimics the redistribution of MIC2 that occurs during attachment and penetration of host cells, and both events are dependent on the actin-myosin cytoskeleton of the parasite. These studies indicate that microneme release is a stimulus-coupled secretion system responsible for releasing adhesins involved in cell attachment.
358 citations
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...Our results differ from a previous report of microneme fusion with rhoptries during host cell invasion by Sarcocystis muris (Entzeroth et al., 1991)....
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TL;DR: During MT or TCT internalization, signal transduction pathways are activated both in the parasite and the target cell, leading to Ca2+ mobilization, and T. cruzi isolate-dependent inhibitory signals, mediated by MT-specific gp90, may be triggeredboth in the host cell and the parasite.
Abstract: Establishment of infection by Trypanosoma cruzi, the agent of Chagasdisease, depends on a series of events involving interactions of diverse parasite molecules with host components. Here we focus on the mechanisms of target cell invasion by metacyclic trypomastigotes (MT) and mammalian tissue culture trypomastigotes (TCT). During MT or TCT internalization, signal transduction pathways are activated both in the parasite and the target cell, leading to Ca 2+ mobilization. For cell adhesion, MT engage surface glycoproteins, such as gp82 and gp35/50, which are Ca 2+ signal-inducing molecules. In T. cruzi isolates that enter host cells in gp82-mediated manner, parasite protein tyrosine kinase as well as phospholipase C are activated, and Ca 2+ is released from IP3-sensitive stores, whereas in T. cruzi isolates that attach to target cells mainly through gp35/50, the signaling pathway involving adenylate cyclase appears to be stimulated, with Ca 2+ release from acidocalciosomes. In addition, T. cruzi isolate-dependent inhibitory signals, mediated by MT-specific gp90, may be triggered both in the host cell and the parasite. The repertoire of TCT molecules implicated in cell invasion includes surface glycoproteins of gp85 family, with members containing binding sites for laminin and cytokeratin 18, enzymes such as cruzipain, trans-sialidase, and an oligopeptidase B that generates a Ca 2+ -agonist from a precursor molecule.
216 citations
Cites background from "Intracellular Ca2+ storage in acido..."
...The Ca2+ required for cell invasion appears to be released from acidocalcisomes, the vacuoles containing a Ca2+/H+ exchange system (Docampo et al. 1995), provided that treatment of MT with a combination of ionomycin plus NH4Cl or nigericin, that releases Ca2+ from these acidic compartments,…...
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